An Interpretive Basis of the Hyperfine Shifts in Cyanide-Inhibited Horseradish Peroxidase Based on the Magnetic Axes and Ligand Tilt. Influence of Substrate Binding and Extensions to Other Peroxidases

Abstract

The 'H NMR hyperfine shift pattern for nonheme resonances in low-spin (S = l/2) cyanide-inhibited horseradish peroxidase, HRP-CN, and cytochrome c peroxidase, CcP-CN, are interpreted in terms of the anisotropy and orientation of the paramagnetic susceptibility tensor in the molecular framework. The orientation of the axes is obtained from a least-squares search for the Euler angles which will rotate the metal-centered crystal coordinates to a coordinate system (the magnetic axes) which quantitatively account for the observed dipolar shifts. Previous 2D NMR has shown that the key active site residues in HRP-CN (Chen, 2.; de Ropp, J. S.; Hernandez, G.; La Mar, G. N. J. Am. Chem. SOC. 1994, 116, 8772) occupy essentially the same position in the active site as in the crystal structure of CcP-CN, such that the crystal coordinates for select protons of CcP-CN could be used for both HRP-CN and CcP-CN NMR analysis. The validity of the magnetic axes description for HRP-CN is supported by the excellent correlation between observed and predicted dipolar shifts, by the ability to predict the shifts for residues not conserved between CcP and HRP, by the quantitative prediction of the influence of substrate binding on the hyperfine shifted pattern in HRP-CN, and by accounting for the significant variation in hyperfine shift pattern among cyanide-inhibited heme peroxidases genetic variants which possess very similar molecular structure. The major magnetic axis is found tilted (-20") from the heme normal in both CcP-CN and HRP-CN, and is similar to that of the Fe-CN tilt in the CcP-CN crystal structure. The effect of binding the substrate benzhydroxaminic acid to HRP-CN is to decrease the tilt of the Fe-CN unit by 9" away from the BHA binding site. The distinct patterns of hyperfine shifted for the cyanide-inhibited lignin and manganese peroxidases indicate that these proteins exhibit significantly less tilt (-5") of the Fe-CN unit from the heme normal than CcP-CN or HRP-CN.

Solution structure of oxidized microsomal rabbit cytochrome b5

“…In early studies, the magnetic anisotropy tensor was determined from pcs by fitting the experimental data to using an available 3D structure of the system [60–65]. Sometimes structural information could be obtained [66–68].…”

Binding mode of benzhydroxamic acid to Arthromyces ramosus peroxidase shown by X‐ray crystallographic analysis of the complex at 1.6 Å resolution

“…Extensive NMR studies have been done to determine the binding mode of aromatic compounds including BHA to HRP [7–12]. Most support that BHA binds to the distal side of the heme extending to the heme iron and is located near the heme edge (pyrrole D) based on observations that BHA interacts strongly with the heme methyl C18H 3 as well as some protons of the distal histidine and arginine residues [11, 12]. In addition, involvement of the distal histidine and arginine residues in BHA binding has been shown by resonance Raman spectroscopy [14].…”

“…Recently, using X‐ray analysis [5], we showed unequivocally the iodide binding site on ARP, a class II peroxidase of the plant peroxidase superfamily [6]. Sufficient spectroscopic data have been accumulated for the aromatic donor molecules to assume that they form complexes with peroxidases [7–12]. In particular, benzhydroxamic acid (BHA, Fig.…”

“…In particular, benzhydroxamic acid (BHA, Fig. 1 ), a competitive inhibitor for the usual donor molecules to horseradish peroxidase (HRP), has been used in studies of the aromatic donor molecule binding site because it forms a tight complex with HRP [7–13]. The NMR study of HRP by Morishima and Ogawa [8]showed that the unique binding site of the aromatic donors is located near the heme edge.…”

“…The NMR study of HRP by Morishima and Ogawa [8]showed that the unique binding site of the aromatic donors is located near the heme edge. Subsequent NMR studies of HRP, including studies using indole‐3‐propionic acid [10]and methyl substituted BHAs [11], have provided more detailed information about the binding modes of aromatic compounds [9, 12]. The hydrogen bonding of BHA with active site residues and the environment of the heme iron also have been investigated for the recombinant HRP‐C and its mutants by resonance Raman spectroscopy [14].…”